The overall aim of the proposed research is to obtain information on the regulation of renal salt and water metabolism (Nephrology). Experiments will be carried out on either isolated perfused or surgically opened cortical collecting tubules (CCTs) of mammalian kidneys. We will study mechanisms of cell homeostasis during changes in reabsorptive Na transport. We will test whether a negative feedback mechanism for Na ions controls the rate of apical Na entry and a positive feedback mechanism controls the rates of K+ exit via apical and basolateral K channels. For this purpose we will measure changes in magnitude and time of [H+]i, [Ca2+]i, [Na+]i, apical and basolateral membrane voltages and ionic conductances during alterations in the rates of apical Na entry and basolateral Na extrusion. [H+]i, [Ca2+]i, and [Na+]i will be measured by BCECF, Fura-2 and SBFI fluorescence techniques, respectively. Membrane voltages and macroscopic electrical conductances will be determined by conventional microelectrode techniques. The effect of induced alterations in [Na+]i, [H+]i and [Ca2+]i on the activity of apical Na and K channels and on basolateral ion channels will be studied in patch clamp experiments. For apical channels surgically opened CCTs will be sued whereas basolateral ion channels will be examined by partial surgical removal of the basement membrane of CCTs, whereby the basolateral cell membrane is exposed for patch clamping. Recently observed basolateral K channels will be characterized in terms of single channel conductance, selectivity, gating response to blocking ions, drugs and metabolites. The role of protein kinase Ca and Ca2+-calmodulin-dependent protein kinase and of prostaglandin in the regulation of ion channels will be evaluated. Because of the importance of intracellular Ca2+ for renal transport of salt and water, we plan to characterize, in a separate project, the molecular properties of the rat renal Na/Ca exchanger. For this purpose, cDNAs encoding the exchanger will be cloned using either the Xenopus oocyte expression system or a rat heart exchanger cDNA probe. Clones will be sequenced and derived amino acid sequences will be analyzed for structural motifs such as hydropathy indices, signal sequences, potential glycosilation sites and local secondary structures. The sequence will be compared with those of other membrane proteins, particularly the cardiac Na/Ca exchanger and Ca channels for common motifs and homologies. Expression of the isolated exchanger cDNA in oocytes permits studies on its functional properties.